This invention relates to methods of identifying transgenic plants and methods of monitoring the segregation of genes in the progeny of transgenic plants.
One of the primary goals of plant genetic research and development is the production of transgenic plants that express a heterologous gene (i.e., produce a protein or an RNA from a gene that normally does not occur in the plant) in an amount sufficient to confer a desired phenotype to the plant. While significant advances have been made in pursuit of this goal, the transformation of heterologous genes into certain plants and the expression of these genes in the plants remains problematic.
In general, plant transformation refers to stably introducing a nucleic acid segment carrying a functional gene (generally, a heterologous gene) into a plant that did not previously contain that gene. In a successful transformation, a DNA construct containing a structural coding sequence is inserted into the genome of a plant by one of several known methods. Examples of known transformation methods include direct gene transfer into protoplasts, microprojectile bombardment, injection into protoplasts, cultured cells and tissues or meristematic tissues, electroporation, and Agrobacterium-mediated transformation.
After effecting delivery of heterologous DNA to recipient cells and plants by any of the methods listed above, identifying the cells and plants exhibiting successful or enhanced expression of a heterologous gene for further culturing and plant regeneration generally occurs by one or more screening methods.
xe2x80x9cScreeningxe2x80x9d generally refers to identifying the cells and/or plants exhibiting expression of a heterologous gene that has been transformed into the plant. Usually, screening is carried out to select successfully transformed seeds (i.e., transgenic seeds) for further cultivation and plant generation (i.e., for the production of transgenic plants). In Arabidopsis thaliana, generally only about 0.5-1% of the seed produced by the transformed plants will contain the heterologous gene of interest. In order to improve the ability to identify transformants, a selectable or screenable marker gene is normally transformed into plant along with the heterologous gene of interest. In such a case, one would then generally assay the potentially transformed cells, seeds or plants by exposing the cells, seeds, plants, or seedlings to a selective agent or agents. For example, transgenic cells, seeds or plants may be screened under selective conditions, such as by growing the seeds or seedlings on media containing selective agents such as antibiotics (e.g., hygromycin, kanamycin, or paromomycin), the successfully transformed plants having been transformed with genes encoding resistance to such selective agents. Alternatively, other methods (e.g., exposing plants transformed with herbicide-resistant genes to herbicides such as BASTA(copyright)) are used to screen the cells, seeds, plants or tissues of the plants for the desired marker gene.
To additionally confirm the presence of the heterologous nucleic acid or xe2x80x9ctransgene(s)xe2x80x9d in the seeds of the transformed plant or in the regenerated plants produced from those seeds, a variety of assays may be performed. Such assays include, for example, molecular biological assays, such as Southern and Northern blotting and PCR; biochemical assays, such as detecting the presence of a protein product, by immunological means (ELISAs and Western blots) or by enzymatic function; and by plant part assays, such as leaf or root assays.
Despite the many methods currently available for screening plants for successful transformation, these methods are not without certain disadvantages. Often, the inclusion of the selective marker gene in the transforming vector places undesirable stress on the plant. Challenging the plants with one or more selective agents further subjects the plant to additional stresses that may be detrimental to the future growth of the plant. For the analysis of unstressed plants, it would necessary to identify transgenic plants using a non-selective marker. Green fluorescent protein (GFP) has been used to screen transformants (see European Patent Application EP 19970924602 assigned to Pioneer Hi-Bred International, Inc.). However, methods of using non-selective markers are presently not widely used, and the development of additional methods of utilizing non-selective markers to screen transformants is desirable.
Methods of detecting successfully transformed plants utilizing nonselective markers would also find use in preliminary screening of primary transformants. In general, primary transformants are not analyzed, because the transformation process can result in multiple insertions (i.e., undesirable mutations). Generally, it is desirable to either out-cross or self-cross the primary transformants to allow the mutations and/or transgenes to segregate. It would be useful to identify primary transformants by selection and then screen for the transformants to be analyzed in the segregating progeny under non-selective conditions.
Furthermore, in smaller plants such as those of the Arabidopsis species (i.e., Arabidopsis thaliana), it is often necessary to bulk seed in order to obtain sufficient material for an analysis of successful transformation. It would be desirable to have a method of identifying transformants in such smaller plants without being required to use methods such as seed bulking in order to obtain sufficient material to analyze transformants.
Finally, it would be useful for breeding purposes to be able to determine, in the F2 population, whether or not a particular plant is homozygous for a particular gene or not. Presently, it is not possible to make such a determination without extensive genetic analyses of the plant.
In view of the foregoing, there is clearly a need for a method of determining whether or not a plant has been successfully transformed without using selective markers, and without having to resort to invasive or destructive assay techniques.
The present inventors have discovered a new method for efficiently, quickly and reliably determining by visual detection whether a particular plant has been successfully transformed with a heterologous gene.
One aspect of the invention is a method for determining the successful transformation of a plant with a heterologous gene. The method is carried out by transforming a first plant with a target DNA construct comprising at least one heterologous gene operatively associated with a morphological marker that encodes a normal phenotypical characteristic of the plant, wherein the plant is homozygous for a mutation of the morphological marker that renders the plant abnormal for the phenotypical characteristic. Seeds are then collected from the first plant and at least one progeny plant grown therefrom. The phenotype of the progeny plant is then detected, wherein the presence of the normal phenotype as indicated by the characteristic encoded by the morphological marker indicates the successful transformation of the progeny plant with the heterologous gene.
An additional aspect of the invention is a method of detecting the successful transformation of a Arabidopsis plant with a heterologous gene. This method of the invention is carried out by transforming a first Arabidopsis plant that is homozygous for a mutation of the GL1 gene with a target DNA construct comprising a heterologous gene operatively associated with the wild-type GL1 gene. The plant is allowed to produce seeds, which are grown into progeny Arabidopsis plants. The presence or absence of trichomes on the progeny Arabidopsis plant is then detected, wherein the presence of trichomes indicates the successful transformation of the progeny plant with the heterologous gene.
Plants that are homozygous for a mutation of a morphological marker that renders the plant abnormal for the phenotypical characteristic further comprising a target DNA construct with at least one heterologous gene operatively associated with a morphological marker that encodes a normal phenotypical characteristic of the plant are also an aspect of the present invention. In a preferred embodiment, the plant is Arabidopsis, and the plant is homozygous for a gl1 mutation.
In addition to providing a method of visually determining whether a particular plant has been successfully transformed with a heterologous gene, the present invention allows the practitioner to track the transgenic state of a plant through successive generations, and track the segregation of transgenes throughout subsequent generations of plant progeny. The methods of the present invention also advantageously allow for the visual determination of the homozygosity, or lack thereof of a transformed plant. The present invention also advantageously allows the practitioner to distinguish between true crosses and self-crosses in plants that have been cross-fertilized.
The foregoing and other aspects of the present invention are explained in detail in the specification set forth below.